a) Field of the Invention
The invention is directed to a device for dispensing and for observing the luminescence of individual specimens in multi-specimen arrangements, particularly for the examination of biological, chemical or cytobiological assays with high specimen throughput (HTS and UHTS). It is suitable in particular for the observation of short-term luminescence phenomena (flash luminescence) directly after dispensing into a plurality of cavities or wells of microtitration plates (microplates) of the type mentioned above.
b) Description of the Related Art
It is particularly important in biochemistry and pharmacology to test as many different substances as possible in the microtitration plates (microplates) within a short period of time by adding reagents or cells. This is usually carried out in the form of an assay in which it is precisely determined at what time the microplate with its specimens must be at what location in what sequence. Usually, the reactions of living cells on substances of pharmacological interest are tested. For this purpose, the cells must be kept in a nutrient medium at a specified temperature and mixed with substances, kept in the incubator again for a defined period of time, etc. But the reverse is also possible, namely, the addition of substances to the wells of the microplate which are charged with reagents or cells.
In many cases, this preparatory handling concludes with the optical measurement of luminescence. For this purpose, one or more reagents are added to the cells before or at the moment of measuring the light. Liquid is added to as many (or all) of the wells of the microplate as possible and, further, the light emission is measured simultaneously starting with the addition of liquid. In this connection, there are many competing demands when high plate throughput is to be achieved with automatic HTS (High Throughput Screening) or UHTS (Ultra-High Throughput Screening).
Since the generated light signals are sometimes expected over only a few seconds, a measurement of intensity with a time resolution in the range of seconds per well is required. However, the total measurement time over an entire microplate should be short.
Due to the high cost of the complex compounds of the dispensing reagents, only a few microliters of a diluted solution may be used on the specimen. This means that a highly sensitive detection system is required (possibly photon counters, e.g., SEV or PMT); conventional video camera systems are not sufficiently sensitive for this purpose.
The reproducible addition of defined amounts of liquid is commonly carried out simultaneously by means of pipetting devices which must be rinsed in the interim in a rinsing station after every pipetting step in order to prevent spreading or entrainment of specimens as a result of immersion in the specimen liquid of the wells. Consequently, all wells of a microplate are pipetted simultaneously for high microplate throughput. This requirement limits the applicability of the device to different types of microplates or requires considerable extra expenditure for changing the pipetting heads.
While a recently introduced contactless method for dispensing (DE 199 11 456 A1) does away with the intermediate rinsing of the dispensing head, it is only applicable to a limited number of dispensing nozzles because of a defined pump control, so that the dispensing head must be displaced by steps relative to the microplate in such a way that the dispensing process can not be accomplished in one step for the entire microplate and the dispensing process is therefore prolonged.
Numerous pipetting devices or dispensing devices are known from the prior art which have an optical observation device for measuring luminescence phenomena resulting from the addition of a reagent in order to be able to detect determined ingredients in the wells of the microplate.
For example, U.S. Pat. No. 4,772,453 describes a device which permits the measurement of luminescence in microplates in that a liquid is added at a fixed time prior to measurement and the measurement is carried out sequentially well by well by means of a photomultiplier (SEV or PMT). The microplate is moved by means of an x-y table. Liquid is injected into the next well to be measured in parallel with the measurement that is taking place.
A disadvantage consists in the large expenditure of time for measurement and in the fact that no simultaneity is achieved between the addition of liquid and the measurement, i.e., an immediate brief light reaction, or flash luminescence as it is called, can not be tracked.
The solution in U.S. Pat. No. 4,366,118 is similarly conceived. This patent likewise describes a sequential readout by PMT. In this case, the addition of substances for light reaction is carried out directly over the measuring position, i.e., simultaneous with the measurement. As in U.S. Pat. No. 4,772,453, the disadvantage consists in the large amount of time spent for measuring well by well in the same way when measuring times in the range of seconds are required.
The European Patent Application EP 0 025 350 describes a luminescence apparatus which allows simultaneous dispensing and measurement. The dispenser comprises a quantity of injection tubes which are moved along with a detector located under every tube in the manner of a fork suspension.
This is disadvantageous in that the detectors must remain below the cups in order to be able to track the time elapsed during a short luminescence phenomenon after dispensing in the wells. It is possible to move to the next column of the microplate only when the measurement has been concluded, i.e., the measurement of a microplate takes a very long time because it is possible to dispense in and measure only one column of the microplate at a time.
WO 01/07896 discloses an apparatus which reads out the luminescence from a microplate from above by means of a cooled CCD camera. Consequently, the addition of liquid is carried out outside the measurement chamber prior to measurement. Further, the optics employ a special Fresnel lens for imaging. The cooled CCD camera which requires long integration times for detecting corresponding intensities of luminescence seems disadvantageous in this case. Individual photons can not be detected. Further, since the camera does not measure at the same time that liquid is added, flash luminescence processes taking place within a few seconds and any fast kinetics over the course of the luminescence can not be recorded.
EP 0 753 734 discloses a device for measuring luminescence accompanied by the addition of liquids. In this case, two different reagents are added in succession with different stationary dispensing units, the microplate being moved below them. Optics for transferring the luminescent light to a video camera are arranged below the microplate in the position of the second dispensing unit. This is disadvantageous in that the first stationary dispensing unit can only be used for adding reagents without luminescence or with luminescence that is not time-critical because the addition of liquid is carried out outside of the measuring area of the camera. Accordingly, the device is only suitable for certain reaction processes. Moreover, (as in EP 0 025 350) measuring and dispensing can not be carried out simultaneously. However, this is not provided probably because the dispensing device has an integrated washing station in which the dispensing tips are rinsed during the ongoing measurement due to the immersion in the liquids of the wells for preventing entrainment of specimens. However, this likewise limits the specimen throughput of an HTS regime.